Tag Archives: Quant-iT DNA BR Kit

Phenol-Chloroform DNA Cleanup – Geoduck gDNA

The gDNA I extracted on 20151104 didn’t look great on the NanoDrop so I decided to perform a phenol-chloroform cleanup to see if I could improve the NanoDrop1000 absorbance spectrum and, in turn, the quality of the gDNA.

  • Added an equal volume (500μL) of phenol:chloroform:isoamyl alcohol (25:24:1) to the DNA
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase to clean tube and discarded interphase & organic phase
  • Added an equal volume (280μL) of chlforoform:isoamyl alcohol (24:1)
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase (210μL) to clean tube
  • Added 0.1vols (21μL) of 3M sodium acetate (pH = 5.2)
  • Added 2vols (420μL) of 100% EtOH
  • Mixed by inversion
  • Incubated @ -20C, 1hr (probably not necessary since gDNA clearly precipitated out as soon as I mixed the sample)
  • Pelleted DNA by centrifuging 15mins, 12,000g, RT
  • Discarded supe
  • Washed pellet with 1000μL cold (-20C) 70% EtOH
  • Centrifuged 5mins, 12,000g, RT
  • Discarded supe
  • Repeated was steps three more times
  • Resuspended pellet in 100μL of Buffer EB (Qiagen)

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 371.83 100 37,183
Quant-IT 100.83 100 10,082

 

The NanoDrop1000 overestimates the concentration of the sample by 3.7x!

Regardless, the yield isn’t all that great (using yield from Quant-IT), which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

The clean up procedure didn’t really seem to help with the geoduck sample, as we’re still seeing a significant amount of absorbance from 230 – 250nm.

Share

Phenol-Chloroform DNA Cleanup – Olympia Oyster gDNA

The gDNA I extracted on 20151104 didn’t look great on the NanoDrop so I decided to perform a phenol-chloroform cleanup to see if I could improve the NanoDrop1000 absorbance spectrum and, in turn, the quality of the gDNA.

  • Added an equal volume (500μL) of phenol:chloroform:isoamyl alcohol (25:24:1) to the DNA
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase to clean tube and discarded interphase & organic phase
  • Added an equal volume (380μL) of chlforoform:isoamyl alcohol (24:1)
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase (320μL) to clean tube
  • Added 0.1vols (32μL) of 3M sodium acetate (pH = 5.2)
  • Added 2vols (640μL) of 100% EtOH
  • Mixed by inversion
  • Incubated @ -20C, 1hr (probably not necessary since gDNA clearly precipitated out as soon as I mixed the sample)
  • Pelleted DNA by centrifuging 15mins, 12,000g, RT
  • Discarded supe
  • Washed pellet with 1000μL cold (-20C) 70% EtOH
  • Centrifuged 5mins, 12,000g, RT
  • Discarded supe
  • Repeated was steps three more times
  • Resuspended pellet in 100μL of Buffer EB (Qiagen)

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

 

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 547.15 200 109,430
Quant-IT 74.26 200 14,851

 

The NanoDrop1000 overestimates the concentration of the sample by 7.4x! That’s really insane!

Regardless, this is a solid yield (using yield from Quant-IT) and, when combined with the other Ostrea lurida gDNA that I isolated today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

The clean up seems to have worked well, as the absorbance spectrum is much improved and nearly mirrors that of the Oly gDNA isolated with the Mollusc Kit.

Share

DNA Isolation – Geoduck Adductor Muscle gDNA

Since we still don’t have sufficient gDNA for the full scope of the genome sequencing, I isolated more gDNA.

Isolated gDNA from 257mg adductor muscle tissue collected by Steven & Brent on 20150811.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 54.93 79 4,339
Quant-IT 34.52 79 2,727

 

The NanoDrop1000 overestimates the concentration of the sample by 1.6x!

Regardless, the yield isn’t all that great, which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

Share

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Since we still don’t have sufficient gDNA for the full scope of the Olympia oyster genome sequencing, I isolated more gDNA.

Isolated gDNA from 118mg outer mantle tissue collected by Steven & Brent on 20150812.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 552.53 79 43,650
Quant-IT 219.07 79 17,307

 

The NanoDrop1000 overestimates the concentration of the sample by 2.5x!

Regardless, this is a solid yield and, when combined with the other Ostrea lurida gDNA that I cleaned up today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

 

Share

DNA Quantification – MBD-enriched Olympia oyster DNA

Quantified the MBD enriched samples prepped over the last two days: MBD enrichment, EtOH precipiation.

Samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

Google Sheet: 20151123_MBD_libraries_quantification

Standard curve looked good – R² = 0.999

MBD recovery ranged from ~250 – 600ng.

MBD percent recoveries ranged from ~2 – 20%. Input DNA quantities were taken from Katherine’s numbers (Google Sheet): Silliman-DNA-Samples

Will contact services about getting bisulfite Illumina sequencing performed.

Share

DNA Quantification – Claire’s C.gigas Sheared DNA

In an attempt to obtain the most accurate measurement of Claire’s sheared, heat shock mantle DNA, I quantified the samples using a third method: fluorescence.

Samples were quantified using the Quant-It DNA BR Kit (Life Technologies/Invitrogen) according the manufacturer’s protocol. Standards were run in triplicate. Due to low sample volumes, only 1μL of each sample was used and was not replicated.

Plate was read on a Perkin Elmer plate reader using the Wallac software. The plate was measured three times, with each well measured for a one second duration on each read.

 

Results:

Spreadsheet: 20150303_gigasHSshearedDNApico

 

 

Comparison of NanoDrop1000, Bioanalyzer, and fluorescence measurements:

Sample NanoDrop (ng/μL) Bioanalyzer (ng/μL) Fluorescence (ng/μL)
2M sheared 48.03 16.28 4.91
4M sheared 190.96 58.52 48.10
6M sheared 141.56 42.98 28.42
2MHS sheared 221.93 32.45 13.48
4MHS sheared 257.48 43.82 11.75
6MHS sheared 202.02 51.12 8.97

 

Not entirely surprising, but the fluorescence method is clearly the most conservative measurement of the three methods. However, I do find the difference between the Bioanalyzer and fluorescence measurements very surprising. I suspected the Bioanalyzer would underestimate the concentrations because I actively selected which peak regions to measure, possibly leaving out some aspect of the sample.

Regardless, will use the most conservative measurements (fluorescence) for decision making.

With our yields, we have insufficient DNA to conduct MeDIP and then subsequent bisulfite conversion and library prep on our own. The recovery from the MeDIP will result in too little input DNA for bisulfite conversion and, in turn, library prep.

However, we do have sufficient quantities of starting DNA (>200ng) for Epigentek’s MeDIP Methyl-seq. I have contacted Epigentek to see if their procedure includes bisulfite conversion after MeDIP (which the website workflow suggests that it does not).

Share

Library Prep – Quantification of C.gigas larvae OA 1000ppm library

The completed BS Illumina library made on Friday (1000ppm) was quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Also quantified Jake’s libraries. Used 1μL of  each sample and the standards.  All standards were run in duplicate.  Due to limited sample, the libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek), using the Gen5 (BioTek) software for all calculations.

Results:

20150209_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The good news is that the standard curve looked fine, with an R²=0.998.

The bad news is that there’s no detectable DNA in the sample, just like last time.

Possibly something is totally shot with this sample?  Will quantify the sheared DNA and decide what to do.

I quantified the sheared DNA and there’s nothing there! Where did it go? I just don’t get it. It was sheared, speed-vac’d and resuspended.  All the DNA should still be in the tubes…

Share

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite library quantification

The two completed BS Illumina libraries (400ppm and 1000ppm) were quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of  each sample and the standards.  All standards were run in triplicate.  Due to limited sample, the two libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek).

 

Results:

The standard curve, raw fluorescence, and calculated concentrations (as determined by the Gen5 (BioTek) software) can be seen here: 20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The standard curve was excellent, exhibiting a R² value = 0.999

 

Sample Concentration (ng/uL)
400ppm 10.592
1000ppm 0.0

 

The 400ppm library looks great, with a good yield.

The 1000ppm library appears to have no measurable quantity of DNA in it.  This is surprising, and disconcerting, as both samples were processed in parallel.  As such, there should be virtually no difference between them, in regards to the library construction process and subsequent yields.

To verify that this wasn’t a pipetting error on my part, I re-quantified the 1000ppm library (in duplicate) and still no detectable DNA.

Will repeat the bisulfite conversion and library construction process on the 1000ppm sample in order to generate a usable library for sequencing.

Share